Laser-scanning microscope with collimator and/or pinhole optics

ABSTRACT

Laser-scanning microscope with at least one detection radiation input, in which an aperture plate is installed in front of the detector, whereby optics with variable transmission lengths and a fixed focal distance is provided for focusing varying wavelengths of the detected light onto the aperture plate level at the detection radiation path, which realizes the imaging from the infinite space into an image level with a finite conjugate distance,  
     and/or  
     with at least one light source launched via an optical fiber, whereby collimator optics with a fixed focal distance, and a variable conjugate distance are down-streamed from the fiber output, which transfers the point source at the fiber output with a numerical aperture into a parallel beam in the infinite space in front of the scan-objective lens, whereby a wavelength-dependent, at least partial compensation of the chromatic distortion of the micro-objective lenses occurs by means of turning the chromatic curve for the illumination wavelength used.

[0001] The laser-scanning microscope (LSM) represents a modem tool for observing smaller structures [1]. As with the traditional microscope, the object is initially imaged via the objective and tube lenses onto the intermediate image level. In a second imaging step of the LSM the confocal principle is realized on the incitation and detection sides. On the incitation side, the launching occurs by means of a punctiform fiber output via a collimator [2], or directly into the infinite space in front of the scanning objective lens, which is focused onto the intermediate image level of the traditional microscope alignment. For the detection the spectrally staggered fluorescent radiation originating from the objective is used, which falls onto a pinhole for detection via the scanning objective and pinhole optics after the intermediate image level. Incitation and detection channels are separated by means of color separators. With the aid of scanning mirrors, which are arranged in the infinite space between the scan objective and collimator/pinhole optics adjacent of the image of the objective pupil, each image field point can be illuminated and detected. The image is then electronically composed of a detector signal and of the information on the scanner positions. The radiation formation occurs in the objective pupil (FIG. 1).

[0002] Due to the fact that optics are often used that correspond to the VIS requirements of traditional light microscopy up to the intermediate image, focal differences occur for the spectral ranges (UV, IR) newly to be developed in dependency of the correction made by the objective and tube lenses. Any systematically occurring fractions can be compensated in the scanning objective lens. Currently, any chromatic distortions beyond this are compensated by a spectral breakdown of the launching and pinhole optics into channels of varying optical path lengths.

[0003] But substantial mechanical adjustment paths often occur even within one channel. This is caused by the mostly strong wavelength dependency of the chromatic distortion within the UV and IR ranges. On the other hand, systems with a high lateral imaging scale (˜100) show a sensitive behavior with regard to the transformation of axial position changes from the object into the image space.

[0004] One solution is the staggering of the entire collimator group that is positioned conjugated to the pinhole level, which can supply a sufficiently large logarithm for the defocusing up to a certain degree [2]. It differs from solutions that are aimed at varying the illumination diameter by means of variooptics [3].

[0005] Literature:

[0006] [1] Wilson, Confocal Microscopy, Academy Press

[0007] [2] Published Application DE 19702753A1

[0008] [3] DE 19654211 A1,

[0009] DE 19901219 A1

[0010] However, the following will describe optics with variable transmission distances, and a fixed focal distance, which realizes an imaging from the infinite space into an image level with a finite conjugate distance. It can be used as pinhole or collimator optics for the refocusing of varying wavelengths at a fixed imaging scale.

[0011] These optics realize the imaging from the “infinite space” into the imaging level, and meet the following requirements:

[0012] 1. fixed focal distance f_(G.)

[0013] 2. large variations of conjugated distances with short adjustment paths, and

[0014] 3. geometrically optical, as well as chromatic correction for ensuring a large spectral bandwidth for beam diameters and wavelengths respectively required for each application

[0015] A solution consisting of at least three elements is recommended, of which the first group has a positive breakout force, the second group has a negative breakout force, and the third group has a positive breakout force (FIGS. 2, 4). The solution is characterized in that in order to change the transmission distance two groups move relative to the opposite of the-remaining group.

[0016] This movement compensates the wavelength-dependent position of the image conjugate distance by means of the movement of optical members in such a way that all wavelengths across the spectral range to be selected can be imaged onto one level. A large variation of the image conjugate distance corresponds to a large focusable spectral range within the image level. The solution allows both the retention of a focal distance, and a large variation of the transmission distance.

[0017] A particularly favorable embodiment of pinhole optics is achieved, if the first and the third groups are moved firmly connected to each other, and the second group is stationary. In paraxial approximation, the achievable conjugate distance change ΔS′_(max) can be estimated at approximately the same focal distances of groups 1 and 3 by means of the correlation ${\Delta \quad S_{\max}^{\prime}} = \frac{\left\lbrack {f_{G}L} \right\rbrack}{\left\lbrack f_{2} \right\rbrack}$

[0018] whereby f_(G) represents the total focal distance of the optics of the overall length L=L₁₂+L₂₃, and f₂ represents the focal distances of the second group.

EMBODIMENT EXAMPLE FOR FOCAL DISTANCE f=160 (PINHOLE OPTICS)

[0019] Focusable pinhole optics with a constant focal distance and pinhole position at low mechanical adjustment movements is intended to be realized with the embodiment f=160. The goal is to optimally adjust the focus for the interested wavelength range per detection radiation input, and to expand this range via LSM5.

[0020] We have chosen the following as the embodiment example:

[0021] The following defines the sequential number:

[0022] 1.: first lens surface L1

[0023] 2.: second lens surface L1

[0024] 3.: first lens surface L2

[0025] 4.: second lens surface L2

[0026] 5.: first lens surface L3:

[0027] 6.: second lens surface L3

[0028] 7.: pinhole

[0029] Distances: d2: thickness lens L1, d3: distance L1-L2, d4:.thickness L2, d5: distance L2-L3, d6: thickness L3, d7: distance L3-pinhole Sequen- Dis- tial tance Radius Glass number d [mm] [mm] Type f′ Lens n₃ ν_(ε) Φ 1 infinite 29,0 N-FK5-L1 59,3 1,48914 70,18  9,0 2  3,0 infinite 3  10,7 43,4 LF5-L2 −30,4   1,58482 40,56  7,0 4  3,5 12,2 5  11,0 −25,3   N-SK2-L3 62,1 1,60994 56,37 11,0 6  3,0 −15,8   7 153,3 infinite

[0030] Examples for Movement:

[0031] The condition of the adjustment according to an existing chromatic distortion is illustrated further below.

[0032] In order to compensate the chromatic distortion of the objective lenses, the following adjustment paths can be realized by means of the mutual staggering of the front and rear lenses as opposed to the stationary center lens. Distance3 [mm] Distance5 [mm] Distance7 [mm] 1.  8.3 13.4 150.9 2. 10.7 11.0 153.3 3.  9.1 12.6 151.7

[0033] The associated dynamics for refocusing is exemplified in FIG. 3a. For evaluating the functionality of the pinhole optics, the circle of confusion on the pinhole level, the chromatic longitudinal aberration, the definition brightness on the pinhole level, and its value with refocusing, as well as the variation of the focal distance across the wavelength, and the corresponding lens position are observed in FIG. 3b. The illustrated lines show the respective sizes when focusing for 390 nm, 546 nm, and 750 nm. It is clear that the chromatic distortion can be compensated by means of the movement of the front and rear lenses, and that the associated definition brightness reaches its best possible refocused value on the pinhole level. The focal distance remains largely constant.

[0034] The chromatic distortion CHL (λ, z₁) of the pinhole optics can be approached by the following expression, particularly with regard to λ₀=546 nm: $\begin{matrix} \begin{matrix} {{{CHL}\left( {\lambda,z_{1}} \right)} = {\sum\limits_{k}{{c_{k}\left( z_{1} \right)}\lambda^{k}}}} \\ {with} \\ {{c_{k}\left( z_{1} \right)} = {q_{k} + {r_{k}z_{1}} + {s_{k}z_{1}^{2}}}} \end{matrix} & (1) \end{matrix}$

[0035] The coordinate z₁ represents the distance3 between the front and center lenses that are used in this instance for characterizing the pinhole optics. As an alternative, the air distances distance5, or distance7, which are hereinafter entitled z₂ or z₃, can be used by means of the combinations

z ₁=20,5 mm−z ₂

z ₁ =z ₃−142,6 mm

[0036] In order to compensate the chromatic distortion of the objective lenses, the following algorithm is recommended:

[0037] The chromatic distortion Δz(λ) of the objective lenses should be calculated into the pinhole levels Δz′(λ)=β²Δz(λ) by using the imaging scale β.

[0038] This chromatic distortion should be compensated by refocusing the pinhole optics for the wavelength λ, i.e.,

Δz′+CHL(λ, z ₁)=0.

[0039] This results in the solutions for z₁, which characterize a suitable adjustment of the movable pinhole optics.

[0040] An assessment of the pre-adjustment setting of the above pinhole optics, characterized by the air distance z₁ between the front and center lenses, can occur as follows:

z ₁=0.16·CHL ₀(λ)+10.5 mm,

[0041] if CHL₀(λ) denotes the chromatic distortion of the objective lens with ideal imaging onto the pinhole level. Furthermore, the following applies:

z ₂=20,5 mm−z ₁

z ₃ =z ₁+142,6 mm

[0042] As the pinhole optics have a free movement range Δz₁ of 21 mm available, chromatic distortions of the objective lenses of up to 130 mm on the pinhole level can be compensated with this arrangement.

6. EMBODIMENT EXAMPLE FOR FOCAL DISTANCE f′=22 (COLLIMATOR OPTICS)

[0043] The collimator optics have the task of transferring the point source at the fiber output with a numeric aperture of about 0.07 into a parallel beam with a diameter of 3.2 mm in the infinite space in front of the scan objective lens. For this purpose, optics with a focal distance of 22 mm is required. It should further realize a partial compensation of the chromatic distortion of the objective lenses by means of turning the chromatic curve. The imaging of infinite into the fiber output can be performed as follows: Sequen- Dis- tial tance Radius Glass number d [mm] [mm] Type f′ Lens n₃ ν_(ε) Φ 1 infinite 10.1 N-FK5 16,4 1,48914 79,5 7,0 2 3,6 −34.0   3 1,7 −11,2   F2 −11,6   1,62408 41,0 7,0 4 3,6 23.2 5 3,7  7.3 N-FK5 16,1 1,48914 79,5 7,0 6 3,6 81.7 7 14,3  infinite

[0044] Distances:

[0045] d2: thickness lens L₁, d3: distance L1-L2, d4: thickness L2, d5: distance L2-L3, d6: thickness L3, d7: distance L3-fiber output

[0046] Adjustments are made so that with a fixed focal distance one fixed wavelength each—particularly λ₀32 546 nm—remains focused, and another wavelength receives a defined chromatic distortion, which serves for the compensation of the chromatic distortion of the objective lenses. The turning of the chromatic curve at a fixed focal distance is realized by means of the movement of the center lens as opposed to the two exterior, firmly connected lenses. filing [mm] Distance3 [mm] Distance5 [mm] Distance7 [mm] at 390 nm 1.7 3.7 14.3 0.17 2.9 2.5 12.0 0.03 5.1 0.3 8.1 −0.18

[0047]FIG. 3a shows the adjustment range of this arrangement. The associated characterization reveals the capability of the collimator group of turning the chromatic distortion by 0.35 mm in the UV range.

[0048] Two sizes are necessary in order to describe the dynamics of the collimator movement. They are the air distance 7 of the last lens at the fiber output z₃, as well as the air distance 5 of the center lens to the last lens z₂. The viewed function assumes a coupled movement z₂ (z₃) in order to focus a wavelength (λ₀=546 nm). The following applies particularly to the breakout force distribution selected above:

z ₂(z ₃)=0.55z ₃−4.2 mm

[0049] The chromatic distortion of the collimator optics can then be expressed as CHL (z₃, λ).

[0050] The (1) applies to the changed development coefficient

c _(k)(z ₃)=q _(k) +r _(k) z ₃

[0051] We want to find such a position z₃, in which the chromatic distortion between the wavelengths λ₁ and λ₂ takes on a defined value in such a way that it is compensated together with that of the objective lenses.

CHL(z ₃,λ₁)−CHL(z ₃,λ₂)=−CHL ₀(λ₁,λ₂)

[0052] From this condition, a position z₃ of the last lens follows opposite of the fiber, as well as the associated position z₂ (z₃) of the center lens, insofar as the respective movement space is sufficient. An assessment of the pre-adjustment of the collimator to the turning of the chromatic curve in such a way that the chromatic distortion CHL₀(λ₂)−CHL ₀(λ₁) caused by the objective lens is compensated on the level of the fiber launching between the two wavelengths λ₁ and λ₂ by means of the collimator at the same focal distance, is given by means of the correlations $\begin{matrix} {z_{2} = {{3.7\quad {mm}} - {0.24\quad {{µm} \cdot \frac{{{CHL}_{O}\left( \lambda_{2} \right)} - {{CHL}_{O}\left( \lambda_{1} \right)}}{\lambda_{2} - \lambda_{1}}}}}} \\ {z_{3} = {{1.8z_{2}} + {7.5\quad {mm}}}} \\ {z_{1} = {{5.4\quad {mm}} - z_{2}}} \end{matrix}$

[0053] The air distance 3 between the front and center lenses z₁ results from the mutual staggering of the front and rear lenses.

[0054] The method for refocusing and turning of the chromatic longitudinal curve with the aid of pinhole optics, as well as of the collimator optics, is particularly advantageous, if

[0055] a) chromatic distortion of the objective lenses dominate,

[0056] b) the system can be refocused (small spherical aberrations, good transmission), and

[0057] c) the contribution of a scan-objective lens for the chromatic distortion can be neglected. 

1. Laser-scanning microscope with at least one detection radiation input, in which an aperture plate is installed in front of the detector, whereby optics with variable transmission lengths and a fixed focal distance is provided for focusing varying wavelengths of the detected light onto the aperture plate level at the detection radiation path, which realizes the imaging from the infinite space into an image level with a finite conjugate distance.
 2. Optics according to claim 1, consisting of at least three members, of which the first group has a positive breakout force, the second group has a negative breakout force, and the third group has a positive breakout force.
 3. Optics according to claims 1 or 2, whereby two groups move relatively opposite of the remaining group for changing the transmission length.
 4. Optics according to one of the claims 1-3 whereby the first and the third groups are moved firmly connected to each other, and the second group is stationary.
 5. Optics according to one of the claims 1-4, whereby the maximum conjugate distance change ΔS′_(max) is determined by the correlation at approximately the same focal distance of the first and third groups, ${\Delta \quad S_{\max}^{\prime}} = {\frac{f_{G}L}{f_{2}}}$

whereby f_(G) represents the total focal distance of the optics of the overall length, L=L₁₂+L₂₃, and f₂ represent the focal distance of the second group.
 6. Optics according to one of the claims 1-5, with the following dimensions and distances: Sequen- Dis- tial tance Radius Glass number d [mm] [mm] Type f′ Lens n₃ ν_(ε) Φ 1 infinite 29,0 N-FK5-L1 59,3 1,48914 70,18 9,0 2  3,0 infinite 5  10,7 43,4 LF5-L2 −30,4   1,58482 40,56 7,0 6  3,5 12,2 5  11,0 −25,3   N-SK2-L3 62,1 1,60994 56,37 11,0  6  3,0 −15,8   8 153,3 infinite


7. Optics according to claim 6, with the following movement of the components: Distance3 [mm] Distance5 [mm] Distance7 [mm]
 1. 8.3 13.4 150.9
 2. 10.7 11.0 153.3
 3. 9.1 12.6 151.7


8. Optics according to one of the claims 1-7, whereby the chromatic distortion CHL (λ, z₁) of the pinhole optics is approached by means of the following expression: $\begin{matrix} {{{CHL}\left( {\lambda,z_{1}} \right)} = {\sum\limits_{k}{{c_{k}\left( z_{1} \right)}\lambda^{k}}}} \\ {with} \\ {{c_{k}\left( z_{1} \right)} = {q_{k} + {r_{k}z_{1}} + {s_{k}z_{1}^{2}}}} \end{matrix}$


9. Optics according to one of the claims 1-8, whereby the compensation of the chromatic distortion of the objective lenses occurs by means of the following method: the chromatic distortion Δz(λ) of the objective lenses is calculated into the pinhole level Δz′(λ)=β²Δz(λ) using the imaging scale β this chromatic distortion is to be compensated by refocusing of the pinhole optics for the wavelength λ, i.e., Δz′+CHL(λ, z ₁)=0 from this, solutions are calculated for the distance between the front and center lenses z₁, which characterize an adjustment of the movable pinhole optics.
 10. Optics according to one of the claims 1-9, whereby an assessment of the pre-adjustment setting of the pinhole optics, characterized by the air distance z₁ between the front and center lenses occurs with: z ₁=0.16·CHL ₀(λ)+10.5 mm, if CHL₀(λ) denotes the chromatic distortion of the objective lens with an ideal imaging into the pinhole level.
 11. Optics according to one of the claims 1-10, whereby the laser-scanning microscope has several detection radiation inputs, whereby at least one detection radiation input can be attached to an interface in the microscope, which has an infinite radiation input.
 12. Laser-scanning microscope, preferably according to one of the previous claims, with at least one light source launched via an optical fiber, whereby collimator optics with a fixed focal distance, and a variable conjugate distance are down-streamed from the fiber output, which transfers the point source at the fiber output with a numerical aperture into a parallel beam in the infinite space in front of the scan-objective lens, whereby a wavelength-dependent, at least partial compensation of the chromatic distortion of the micro-objective lenses occurs by means of turning the chromatic curve for the illumination wavelength used.
 13. Optics according to claim 12, consisting of at least three members, of which the first group has a positive breakout force, the second group has a negative breakout force, and the third group has a positive breakout force.
 14. Optics according to one of the claims 12-13, whereby the turning of the chromatic curve is realized at-a fixed focal distance by means of the mutual movement of the exterior two lenses, and the center lens.
 15. Optics according to one of the claims 12-14, whereby the optics transfer the point source at the fiber output with a numerical aperture from 0.07 into a parallel beam with the diameter of 3.2 mm in the infinite space in front of the scan-objective lens.
 16. Optics according to one of the claims 12-15, whereby the collimator optics have a focal distance of about 22 mm.
 17. Optics according to one of the claims 12-16, with the following dimensions and distances: Sequen- Dis- tial tance Radius Glass number d [mm] [mm] Type f′ Lens n₃ ν_(ε) Φ 1 infinite 10.1 N-FK5 16,4 1,48914 79,5 7,0 2 3,6 −34.0 3 1,7 −11.2 F2 −11,6   1,62408 41,0 7,0 4 3,6 23.2 5 3,7 7.3 N-FK5 16,1 1,48914 79,5 7,0 6 3,6 81.7 7 14,3  infinite


18. Optics according to claim 17, Whereby the turning of the chromatic curve at a fixed focal distance is realized by means of the movement of the center lens opposite of the two exterior, firmly connected lenses as follows: Filing [mm] Distance3 [mm] Distance5 [mm] Distance7 [mm] at 390 nm 1.7 3.7 14.3 0.17 2.9 2.5 12.0 0.03 5.1 0.3 8.1 −0.18


19. Optics according to one of the claims 12-18, whereby a coupled movement z₂ (z₃) of the second and the third lenses occurs in order to focus a wavelength, whereby z ₂(z ₃)=0.55z ₃−4.2 mm is with Z2 : air distance of the center lens to the last lens opposite of the illumination direction, and Z3 : air distance of the last lens to the fiber output.
 20. Optics according to one of the claims 12-19, whereby the chromatic distortion CHL₀(λ₂)−CHL₀(λ₁) caused by the objective lens is compensated on the level of the fiber launching between the two wavelengths λ₁ and λ₂ by means of the collimator at the same focal distance, is given by means of the correlations $\begin{matrix} {z_{2} = {{3.7\quad {mm}} - {0.24\quad {{µm} \cdot \frac{{{CHL}_{O}\left( \lambda_{2} \right)} - {{CHL}_{O}\left( \lambda_{1} \right)}}{\lambda_{2} - \lambda_{1}}}}}} \\ {z_{3} = {{1.8z_{2}} + {7.5\quad {mm}}}} \\ {z_{1} = {{5.4\quad {mm}} - z_{2}}} \end{matrix}$ 